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Equilibrium and Kinetic Constants for the Thiol-Disulfide Interchange

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Equilibrium and Kinetic Constants for the Thiol-Disulfide Interchange Proc. Natl. Acad. Sci. USA Vol. 89, pp. 7944-7948, September 1992 Biochemistry Equilibrium and kinetic constants for the thiol-disulfide interchange reaction between glutathione and dithiothreitol (disulfide reduction/thiol oxidation/redox potential) DAVID M. ROTHWARF AND HAROLD A. SCHERAGA* Baker Laboratory of Chemistry, Cornell University, Ithaca, NY 14853-1301 Contributed by Harold A. Scheraga, June 15, 1992 ABSTRACT The equilibrium and rate constants for the KObs [DTToX] [GSH]2 reaction between oxidized and reduced glutathione and oxi- eq [Drrred] [GSSG]' [2] dized and reduced dithiothreitol have been determined at several pH values and temperatures. The measurements in- where the brackets indicate concentrations at equilibrium. volve approach to equilibrium from both directions, quenching Unfortunately, a wide range of values has been reported of the reaction by lowering the pH or by addition of methyl for this thiol-disulfide exchange equilibrium constant. Values methanethiosulfonate, separation of reactants and products by of 8800 M at pH 7 and 300C (10) and 13,000 M at pH 7 and an reverse-phase HPLC, and determination of their concentra- unspecified temperature (11) have been reported, based on an tions. Analysis of reaction mixtures was carried out at various indirect technique involving a lipoamide dehydrogenase- times to assure that equilibrium had been reached and to mediated reaction between NAD+ and lipoamide. Use of determine kinetic constants prior to the attainment of equilib- intermediates populated during the regeneration of bovine rium. pancreatic trypsin inhibitor (BPTI) led to a value of 1200 M at pH 8.7 and 250C (12). A direct HPLC method, very similar The thiol-disulfide interchange reaction is important in a to the technique to be reported here, gave values of -200 M variety of biological systems (1-3) and, in particular, in at pH 7 and 8, 250C, and 380 M at pH 8.7, 250C (13). In the studies of the regeneration of disuffide-containing proteins direct method (13), the equilibrium was approached from from their reduced forms (4-6). The thiol/disulfide reagents only one direction and insufficient experimental details were in widest use in studies of the regeneration of proteins are provided to resolve the controversy. Our own indirect mea- oxidized glutathione (GSSG) and reduced glutathione (GSH), surements (unpublished results), obtained while studying the primarily because they are known to occur at significant regeneration of ribonuclease A, resulted in a value of 260 M concentrations in biological systems (7) and because they at pH 8, 25°C, and are in agreement with those of Chau and exhibit a suitable redox potential. Nelson (13). The oxidation of monothiols such as GSH is a bimolecular While it will not be discussed in detail here, the value ofthis process and is entropically different from the unimolecular equilibrium constant and the rate of reduction of GSSG by process of disulfide formation that occurs in proteins. In dithiothreitol (DTTmd) are essential for explaining the path- contrast, the formation of cyclic disulfides such as oxidized ways ofregeneration ofribonuclease A and for understanding dithiothreitol (DTToX) results from a unimolecular process. the general problem of how native protein structures regen- Glutathione forms stable mixed disulfides with protein thiols, erate. The purpose of this paper is to present experimental which greatly complicates studies of regeneration pathways. details of the direct method, sufficient to confirm the accu- On the other hand, while DTTOX is a much weaker oxidizing racy of the results of Chau and Nelson (13) at pH 8 and 25°C, agent, it does not form stable mixed disulfides (because the which are consistent with our data from the regeneration of cyclization reaction is very fast), making it a useful reagent ribonuclease A, and to determine the values of the equilib- for studies of protein regeneration (5, 8). It has been argued, rium constants and rate constants under the solution condi- though incorrectly, that, while these reagents differ in their tions that are directly relevant to our work on the regener- entropies of disulfide formation, they are similar in their ation of ribonuclease A (8). We have also carried out equil- abilities to regenerate protein when concentrations are ibrations under other conditions (pH 7, 30°C) to permit direct adjusted to give similar redox potentials (9). comparison with the values for the equilibrium and kinetic The role that these different types (mono- and cyclic thiol) constants determined by Szajewski and Whitesides (10). of reagents play is a key feature of the detailed regeneration processes of proteins. Any attempt to distinguish these different roles requires a quantitative determination of their MATERIALS AND METHODS relative redox potentials. In order to make comparisons Materials. Ultrapure DTTred was obtained from Boeh- between the results ofregeneration experiments using dithio- ringer Mannheim and recrystallized from absolute ether. threitol and those using glutathione, it is necessary to know DTTOX (Sigma) was purified by the method ofCreighton (14). the equilibrium constant for the reaction between glutathione GSH and GSSG were obtained from Boehringer Mannheim and dithiothreitol. The equations describing this equilibrium and were purified by reverse-phase HPLC on a 5-pm C18 are Dynamax column (Rainin, Woburn, MA) with 0.09%o tri- fluoroacetic acid (TFA) in water isocratically as the mobile GSSG + DTTred ; 2GSH + DTfoX [1] Abbreviations: DTTOX, oxidized dithiothreitol; DTTed, dithiothrei- tol; GSH, reduced glutathione; GSSG, oxidized glutathione; BPTI, The publication costs of this article were defrayed in part by page charge bovine pancreatic trypsin inhibitor; DDS, disulfide detection system; payment. This article must therefore be hereby marked "advertisement" MMTS, methyl methanethiosulfonate; TFA, trifluoroacetic acid. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 7944 Downloaded by guest on September 24, 2021 Biochemistry: Rothwarf and Scheraga Proc. Natl. Acad. Sci. USA 89 (1992) 7945 phase. All other reagents were of the highest grade commer- alent volume of a TFA solution sufficient to lower the pH to cially available. 2. The concentration of TFA used was in the range 150-300 Purity. Because the equilibrium constant of Eq. 2 is mM. In other experiments, a 25-fold volume of 25 mM TFA expected to be large and has a molar concentration depen- was used to lower the pH to 2. There was no difference within dence, the concentrations of GSH and DTTYX at equilibrium experimental error between the two techniques. While it is will be as much as 4 orders of magnitude greater than those unlikely that different acid concentrations during quenching of GSSG and DTTmd under the conditions used here. There- could yield the same equilibrium constant if shuffling oc- fore, even a small level of impurity could lead to erroneous curred during the quenching, it is not a definitive control. results if it were coeluted with DTTr"d or GSSG. All reagents Therefore, an additional method was used to quench some of were, therefore, purified to >99.9% purity as judged by the samples. A 15-fold molar excess of methyl methanethio- HPLC at 210 nm. In addition, the GSSG and GSH were sulfonate (MMTS) over free thiol was added in a 3:1 volume checked by NMR on a Varian XL-400 instrument prior to ratio at pH 8.0. After 5 min, the blocking reaction was purification, because we have observed that glutathiones quenched with 0.5 M TFA to pH 2 prior to HPLC analysis. obtained from some suppliers contained significant amounts Within experimental error, there was no difference between (in some cases >20%) of a-glutamylcysteinylglycine (M. the results from any of the quenching procedures. In control Adler, D.M.R., and H.A.S., unpublished results) as well as experiments on mixtures of DTTJX and DTTred, the MMTS cysteinylglycine (T. W. Thannhauser and H.A.S., unpub- quenching conditions blocked >95% of the DTTred com- lished results). Significant heterogeneity in the GSSG and pletely (the remainder presumably cyclized to DTTOX before GSH may be the origin of the wide range of values reported the second thiol of DTTred was blocked). for the dithiothreitol-glutathione equilibrium constant. NMR Fractionation of Species. After quenching, all equilibration spectra of the materials used in these studies revealed only were on a Waters radial the presence of GSSG and GSH. mixtures fractionated Nova-Pak C18 The high purity of the starting materials, however, is an compression column. A Spectra-Physics SP8800 gradient inadequate control to exclude experimental errors due to pump and a Gilson UV116 detector set to 210 nm constituted contamination, since the formation of small amounts of the delivery and detection system. A binary gradient using degraded starting material could also complicate the inter- solvent systems A (0.09% TFA/water) and B (0.09%o TFA/ pretation of the data if they were coeluted with DTTred or acetonitrile) was run at a flow rate of 1 ml/min. The com- GSSG. Therefore, we measured the equilibrium constant position of the mobile phase was held at 100% A for 10 min, over a >10-fold range of the starting thiol and disulfide and then a linear gradient to 15% B in 30 min was imple- concentrations. Since the equilibrium constant has a molar mented. This gradient was used in all subsequent HPLC concentration dependence, a 10-fold increase in the concen- analyses described below. All data were digitized and stored trations of both GSH and DTTYX will lead to an -1000-fold on a Prime 750 computer. A typical chromatogram is shown increase in the product of the concentrations of GSSG and in Fig. 1. DTTred. Since it is the concentrations of GSSG and DTTred Relative Extinction Coefficients. Given the form of the that are sensitive to the level of impurities, and since they equilibrium expression as shown in Eq.
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